匍枝根霉cbh2基因的克隆表达与结构分析

Cloning,expression and structure analysis of cbh2gene from Rhizopus stolonifer

采用反向PCR技术从匍枝根霉中克隆cbh2基因,该基因编码440个氨基酸的蛋白质。利用DS2.5软件进行同源模拟和建立分子动力学模型,研究cbh2催化纤维六糖水解过程中结合区（CBM）上的关键位点,催化区（CD）上催化隧道关键位点的相互依赖作用,水解β-1,4糖苷键,生成纤维二糖。把cbh2基因与p ET22-b（＋）载体连接,实现了在E.coli BL21（DE3）中原核表达。通过镍柱层析、DEAE FF层析和G-75层析三步纯化法获得46 k Da的酶蛋白,比活力为4.67 IU/mg。纯化的重组酶具有高水平的催化活性,对微晶纤维素具有较好的水解特性,CBHII酶的Km为7.358（mg/m L）。研究CBHII酶的基本特性,其最适反应温度为50℃,最适反应p H为5.0,CBHII酶在p H 4～7下具有较宽的稳定性,在65℃以下热稳定性较好,在催化过程中保持较高的活力。

The cbh2 gene encoding a protein of 440 amino acids was cloned from Rhizopus stolonifer by inversePCR technology. Homology model and molecular dynamics model were established by DS2. 5 software to study CBHII catalytic hydrolysis process of cellohexose on the cellulose-binding modules（ CBM） and the catalytic domain（ CD） of key sites,which hydrolyzed β-1,4 glycosidic bond and generated cellobiose. The gene was inserted into the expression vector p ET22-b（ ＋） and transformed into E. coli BL21（ DE3）. Using a three-step column chromatography procedure of Ni affinity chromatography,DEAE FF chromatography and G-75 chromatography,the purified enzyme with molecular weight of about 45 k Da and specific activity of 4. 67 IU / mg was obtained. The purified recombinant enzyme showed high activity towards microcrystalline cellulose,and CBHII Kmwas 7. 358 mg / m L. And the basic characteristics of CBHII were also analyzed. The optimal conditions for the enzymatic reaction were about p H 5. 0 at 50 ℃. At p H 4 ～ 7 and below 65 ℃,the enzyme showed high activity and it retained most of activity in the catalytic process.